Enhancer of zeste 2 (EZH2), a histone methyl transferase subunit of the Polycomb Repressive Complex 2 (PRC2), catalyzes the methylation of lysine residue 27 on histone 3 (H3K27). Methylation of H3K27 inhibits the transcription of nearby genes by blocking the recruitment of the proteins necessary for gene activation. The PRC2 complex and the histone modifications that it regulates are tightly controlled in normal cells but can become dysregulated in cancer to aberrantly activate or repress gene expression. Over the last decade, many independent studies have established that EZH2 is highly expressed in numerous cancers and recurrently mutated in several others. Overexpression of EZH2 is associated with aggressive progression and poor prognosis. We recently demonstrated that overexpression of EZH2 is causative of lung adenocarcinoma in mice. Likewise, activating somatic mutations of EZH2 have been identified in B cell lymphoma and malignant melanoma. We developed mouse models to express the gain-of-function mutant of EZH2 in B cells or melanocytes, which caused high-penetrance lymphoma or melanoma, respectively. Our studies with mouse models that express the gain-of-function mutant of EZH2 suggest that EZH2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure inducing both repression and activation of PRC2-regulated loci. The extensive evidence linking EZH2 activity to cancer has prompted interest in the underlying biological mechanisms, including the link to the tumor immune environment. As such, we developed a novel EZH2 small molecule inhibitor, JQEZ5, that can efficiently block the enzymatic function of both wild-type and mutant EZH2 and reduce the methylation of H3K27. The inhibitory activity of JQEZ5 impedes the growth of cancer cells in culture and in mouse models of lung cancer, lymphoma and melanoma. Mechanistically, JQEZ5 is a non-covalent inhibitor that competes off natural co-factor S-adenosine methionine (SAM) from binding with EZH2. The SAM-competitive nature of all current EZH2 inhibitors largely limited their in vivo potency, which could potentially limit their usage in cancer therapy. To improve the in vivo efficacy of EZH2 inhibitors and reduce off-target toxicity, we propose to develop and characterize a new class of covalent EZH2 inhibitors that can irreversibly bind to unique cysteine residue (C663) present in human and mouse EZH2. We hypothesize that the irreversible biding of this new class inhibitors can overcome key limitations of the existing non-covalent inhibitors, and improve in vivo potency and selectivity. We will employ the new covalent EZH2 inhibitors to further investigate cancer biology of EZH2 inhibition in lung cancer, including the immunomodulatory effects of EZH2 inhibition. We will utilize our cell culture and mouse models of EZH2-driven cancer to explore the impact of irreversible inhibition of EZH2 on gene expression and the tumor immune microenvironment while driving hit-to-lead optimization of covalent EZH2 inhibitors that can prompt clinical investigation to fully explore the translational potential of EZH2 inhibitors.